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Endocrinology Vol. 139, No. 10 4155-4163
Copyright © 1998 by The Endocrine Society


ARTICLES

A Protective Role for Heme Oxygenase Expression in Pancreatic Islets Exposed to Interleukin-1ß1

Jing Ye2 and Suzanne G. Laychock

Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, the State University of New York, Buffalo, New York 14214

Address all correspondence and requests for reprints to: Dr. S. Laychock, 102 Farber Hall, the State University of New York at Buffalo, School of Medicine, Buffalo, New York 14214. E-mail: laychock{at}acsu.buffalo.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Heme oxygenase (HO)-1 expression was investigated in rat isolated pancreatic islets. Freshly isolated islets showed no evidence of HO-1 expression. After a 20-h culture, there was a small increase in HO-1 in control islets, and interleukin-1ß (IL-1ß) induced HO-1 expression above control levels. NG-monomethyl-L-arginine inhibited the IL-1ß-induced increase in HO-1. Sodium nitroprusside-generated nitric oxide also increased HO-1 expression. CoCl2 induced a concentration- and time-dependent increase in HO-1, but not heat shock protein 70, expression. Cobalt chloride (CoCl2) protected islets from the inhibitory effects of IL-1ß on glucose-stimulated insulin release and glucose oxidation. Nickel chloride did not mimic the effects of CoCl2. An inhibitor of HO-1 activity, zinc-protoporphyrin IX (ZnPP), prevented the protective effect of CoCl2 on insulin release with IL-1ß but did not affect HO-1 expression or the inhibitory response to IL-1ß alone. ZnPP also inhibited the protective effect of hemin in IL-1ß-treated islets. CoCl2 inhibited the marked increase in islet nitrite production in response to IL-1ß. Cobalt-protoporphyrin IX (CoPP), which increased HO expression and activity, also protected islets from the inhibitory effects of IL-1ß, even though IL-1ß largely blocked the CoPP-induced increase in HO-1 expression. In ßHC9 cells, CoCl2 increased HO-1 expression and HO activity, whereas CoPP directly activated HO. ZnPP inhibited basal and CoCl2-stimulated HO activity. Thus, increased HO-1 expression and/or HO activity in response to CoCl2, CoPP, and hemin, seems to mediate protective responses of pancreatic islets against IL-1ß. HO-1 may be protective of ß-cells because of the scavenging of free heme, the antioxidant effects of the end-product bilirubin, or the generation of carbon monoxide, which might have insulin secretion-promoting effects and inhibitory effects on nitric oxide synthase.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HEME oxygenase (HO) [heme, hydrogen-donor:oxygen oxidoreductase ({alpha}-methene-oxidizing, hydroxylating), EC 1.14.99.3], also known as heat shock protein (hsp)32, is a membrane-bound enzyme responsible for catalyzing heme degradation. HO uses dioxygen and nicotinamide-adenine dinucleotide phosphate as cofactors, with the resulting products of the reaction being carbon monoxide, iron, and biliverdin (1). Biliverdin is converted to bilirubin by a ubiquitous cytosolic enzyme biliverdin reductase (2). HO exists in two forms as products of distinct genes: HO-2 (36 kDa) is constitutive, and HO-1 (32 kDa) is inducible. Both HO-2 and an inducible HO-1 have been identified in rat pancreatic islets (3, 4, 5), as well as other tissues (6). HO-1 expression increases in response to heme and stressors such as UV radiation and oxidative stress, as well as endotoxin, hormones, and heavy metals (6). HO-1 induction may protect cells by reducing heme levels that catalyze oxygen radical reactions and elevating bilirubin, which has antioxidant properties (1). Bilirubin inhibits autoxidation or peroxyl-radical-induced oxidation of unsaturated fatty acids, apparently through peroxyl radical-trapping antioxidant abilities (7, 8). In addition, bilirubin scavenges and quenches toxic singlet oxygen (9). The expression of HO-1 is regulated by the family of AP-1 transcription factors, among others, and the expression and DNA binding activity of c-Fos and c-Jun are stimulated by prooxidants such as heavy metals, hydrogen peroxide, and UV-irradiation (10, 11).

Pancreatic islets respond to stress through the induction and activation of several stress-activated proteins. Interleukin-1ß (IL-1ß) induces an inflammatory response in pancreatic islets, characterized by increases in inducible nitric oxide synthase (iNOS) levels and increased nitric oxide (NO)/nitrite levels (12, 13, 14, 15). IL-1ß and heat shock increase expression of hsp70 (16, 17), as well as HO-1 (3, 18). A protective effect of heat shock on islet cells may be associated with reduced lysis from NO, reactive oxygen intermediates, and streptozotocin (17); but the response is nonspecific because many hsp respond to this stimulus. On the other hand, liposomal delivery of hsp70 into islet cells protected the cells from IL-1ß effects on insulin secretion (19), suggesting that heightened levels of specific hsp can protect ß-cells from inhibitory effects of the cytokine. Hemin, which increases HO-1, has also been found to partly counteract the IL-1ß inhibition of insulin release and to protect against IL-1ß-induced inhibition of aconitase activity and glucose oxidation (18), perhaps through antioxidant mechanisms. However, hemin has also been reported to increase insulin and glucagon secretion from normal rat islets (4). The present study investigates the potential for a heavy metal to induce the synthesis of HO-1 and affect pancreatic ß-cell responses to IL-1ß.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Materials
D-[U-14C]glucose (250–360 mCi/mmol) was from American Radiolabeled Chemicals (St. Louis, MO). [125I]Insulin (human) was from New England Nuclear-DuPont (Wilmington, DE). CMRL-1066 medium was from Life Technologies (Grand Island, NY). Recombinant human IL-1ß was from R&D Systems (Minneapolis, MN); ED50 in cell proliferation assay is 5–10 pg/ml. NG-monomethyl-L-arginine (NMMA) was purchased from Calbiochem (San Diego, CA). Rat insulin for RIA standard was a gift from Eli Lilly Co. (Indianapolis, IN). Antisera to HO-1 and hsp70 was from Stressgen Biotechnologies Corp. (Victoria, British Columbia). Kodak AR-10 x-ray film was from Eastman Kodak Co. (Rochester, NY). All other reagents were from commercially available sources.

Tissue isolation/culture
Isolated pancreatic islets were prepared from male rats and were cultured for 20 h in CMRL-1066 medium containing 5.5 mM glucose, as described previously (20). Other agents present during culture are specified in the text. All animal procedures were approved by the Institutional Animal Care and Use Committee. When sodium nitroprusside (SNP) was used to generate nitric oxide, cyanide ions were inactivated by rhodanese (1.3 U) and sodium thiosulfate (25 µM) (21).

ßHC9 insulinoma cells, from an established line derived from hyperplastic pancreatic islets of transgenic mice harboring SV40 large T-antigen in ß-cells (22), were a gift from Dr. D. Hanahan. The cells were cultured in complete DMEM medium, deficient in pyruvate and containing 10% bovine FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml), at 5% CO2-95% air, 35 C.

Glucose oxidation
Glucose oxidation was determined by quantitation of picomoles of glucose oxidized to 14CO2, based upon the specific activity of [U-14C]glucose (17 mM), as described previously (23).

Insulin release
Isolated islets (10/sample) were cultured 20 h in CMRL-1066 medium in the absence or presence of IL-1ß (1 ng/ml) and other agents, as indicated in the text. Cultured islets were subsequently washed to remove culture medium, serum, and experimental agents. Washed islets were preincubated for 1 h in Krebs Ringer bicarbonate (KRB) buffer (pH 7.4), containing 5.5 mM glucose, 0.01% BSA, and HEPES (16 mM) but lacking IL-1ß or the other agents present during the overnight culture, as described previously (23). After preincubation, the islets were placed in fresh KRB buffer, and an aliquot was removed to determine zero-time insulin levels. Islets were incubated at 5.5 or 17 mM glucose for 60 min, after which an aliquot of the incubation buffer was removed to determine insulin release. Insulin was quantitated by RIA, and zero-time insulin levels were subtracted from 60 min values. Insulin in culture medium was also quantitated after the overnight culture. Total islet insulin content is the amount of insulin extracted from islets, using ethanol (70%) in HCl (1 N), plus the insulin released during static incubation.

Nitrite measurements
NO synthesis was estimated by the accumulation of total nitrate and nitrite in culture medium of islets after an 18-h culture, as described previously (23).

Western blot analysis
Isolated islets were cultured, as described above, and sonicated in deionized water (0.1 ml) containing leupeptin (5 µg/ml), aprotinin (76 µg/ml), and pepstatin (1 µg/ml), at 4 C. Islet protein levels were determined by Bio-Rad protein assay using BSA as standard. The remaining islet protein was precipitated using a 10x volume of ice-cold acetone and microcentrifugation at 14,000 rpm for 5 min; protein recovery was 94% using BSA as standard. The protein was resuspended in SDS sample mix (0.062 M Tris-HCl, 1% ß-mercaptoethanol, and 2% SDS) containing protease inhibitors, as specified above, and boiled for 5 min. Proteins were separated by SDS/15% PAGE (24). Equal amounts of protein per sample were used in each experiment. Proteins were electrophoretically transferred to nitrocellulose membranes and reacted with rabbit antirat HO-1 (1:1250 dilution) or antimouse hsp70 (1:500 dilution), followed by horseradish peroxidase-conjugated goat antirabbit or antimouse IgG. Detection of HO-1 was by the enhanced chemiluminescence method and Kodak AR-10 X-ray film exposure. Each experimental determination was repeated at least two or three times. Densitometric analysis of each band on Western blot was analyzed by Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA). The data for each experiment were normalized to control values by subtracting the image density of control HO-1 levels from each experimental sample for quantitative analysis. Values for densitometric analysis are expressed as relative density units (RDU).

Heme oxygenase (HO) activity
Total HO activity was quantitated by the generation of bilirubin from heme in cell homogenates, essentially as described previously (25). Treated and untreated ßHC9 cells were incubated for 18 h; and then the cells were collected, washed with PBS, and suspended in MgCl2 (2 mM) phosphate (100 mM) buffer (pH 7.4). The cells were frozen and thawed three times, sonicated, and centrifuged at 5000 rpm for 5 min in a microfuge at 4 C. An aliquot of the supernatant (0.3–0.4 mg protein) was added to the NADPH-generating system (0.4 ml) containing NADPH (0.8 mM), glucose-6-phosphate (2 mM), glucose-6-phosphate dehydrogenase (0.2 U), potassium phosphate buffer (100 mM, pH 7.4), hemin (10 µM), and 2 mg protein from rat liver (35,000 rpm supernatant fraction) as a source of biliverdin reductase, and allowed to incubate for 2 h at 37 C in amber tubes. The reaction was stopped, and bilirubin was extracted by addition of 0.8 ml chloroform; the aqueous layer was extracted twice with additional chloroform. The bilirubin extract was dried and resuspended in 0.5 ml chloroform for determination of bilirubin by the difference in absorption at 464 nm and 530 nm. Readings for samples, prepared in the absence of cell homogenate (blank), were subtracted from all other values prepared in duplicate. HO activity is expressed as pmol bilirubin produced/mg protein·2 h.

Determination of zinc-protoporphyrin IX (ZnPP) uptake by cells
ßHC9 cells were cultured 20 h with ZnPP (10 µM). Then, the cells were collected, washed with PBS three times, and resuspended in formic acid (88%), as described previously (25). The spectrophotometric absorbance was read at 407 nm, and cell extract absorbance was compared with ZnPP standard to quantitate uptake.

Statistical analysis
The data are presented as the mean ± SE and were analyzed by one-way ANOVA combined with the post hoc Student/Newman-Keuls multiple-comparison test; P < 0.05 was accepted as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cobalt effects on islet HO-1
In freshly isolated islets, HO-1 levels were not detectable on immunoblot. However, after culture for 20 h at a basal concentration of glucose (5.5 mM), islet HO-1 levels were detectable (Fig. 1AGo). Culture of rat islets has been previously reported to increase HO expression (5), perhaps through accumulation of glucagon (6) or factors present in the culture medium/serum. Islet culture with IL-1ß also induced a small increase in HO-1 levels, which were slightly but consistently above control values (Figs. 1AGo and 2Go). The presence of cobalt chloride (CoCl2) during islet culture induced a high level of expression of HO-1, which was concentration-dependent from 1–100 µM (Figs. 1BGo and 2Go). In contrast, the presence of similar concentrations of nickel chloride (NiCl2) in the islet culture did not induce comparable changes in HO-1 levels, compared with CoCl2 (Fig. 1AGo). CoCl2, in combination with IL-1ß, also enhanced HO-1 levels above those observed with the cytokine alone (Figs. 1AGo and 2Go), whereas NiCl2 did not affect IL-1ß-induced HO-1 levels (data not shown). However, islet HO-1 levels in the presence of IL-1ß and CoCl2 (10 µM) were significantly lower than in the presence of CoCl2 alone, perhaps because of cytokine effects on protein synthesis. A time-course analysis showed that CoCl2 induced an increase in islet HO-1 levels within 8 h, and IL-1ß induced an increase after 18 h of culture (Fig. 3Go, A and B). When both CoCl2 and IL-1ß were present together in islet culture, HO-1 levels were increased within 4 h, and much higher levels were observed within 8 h, compared with islets treated with IL-1ß alone (Fig. 3CGo). In comparison with HO-1, islet hsp70 levels did not seem to be affected during islet culture with CoCl2 or with the concentration of IL-1ß used in this study (Fig. 3Go, A and B).



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Figure 1. Immunoblot analysis of HO-1 expression in isolated islets. Islets (A and B) or ßHC9 cells (C) were cultured 20 h in the presence and/or absence of IL-1ß (1 ng/ml), CoCl2 (1, 10, or 100 µM; Co1, Co10, or Co100, respectively), NiCl2 (10 or 100 µM; Ni10 or Ni100, respectively), as indicated. Islet or cell proteins were separated by SDS/15% PAGE, and HO-1 (32 kDa) was detected by immunoblot. Results are for three representative experiments.

 


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Figure 2. Islet HO-1 expression levels. Islets were cultured 20 h in the absence (basal) or presence of IL-1ß (1 ng/ml), NMMA, CoCl2 (Co), or CoPP at the concentrations (µM) indicated. HO-1 expression on immunoblot was determined in RDU, and data were normalized by subtraction of basal values in paired groups. Values are the mean ± SE for normalized RDU in three independent experiments. Significant differences were determined by one-way ANOVA and Student/Newman-Keuls multiple-comparison test. *, P < 0.05 vs. IL-1ß-treated control islets.

 


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Figure 3. Islet time-dependent HO-1 and hsp70 expression levels. Islets were cultured for up to 18 h in the absence (control) or presence of CoCl2 (10 µM) (Co10) and/or IL-1ß (1 ng/ml), as indicated. HO-1 (32 kDa) and hsp70 (70 kDa) levels were determined by immunoblot with 5 µg islet protein per lane. Results are representative of three experiments.

 
Culture of islets with the NO-generating compound, SNP, in the presence of rhodanese and Na2S2O3, to scavenge the cyanide component of the SNP (21), also increased HO-1 expression. The increase in HO-1, in response to SNP, was both concentration- and time-dependent (Fig. 4Go, A and B). The maximum concentration of SNP inducing HO-1 expression was 100 µM, and a higher concentration (250 µM) seemed to be less effective, perhaps because of toxic effects on the cells. To determine whether NO production mediated the effect of IL-1ß on HO-1 expression, an inhibitor of nitric oxide synthase, NMMA, was included during the islet culture with IL-1ß. Western blot analysis revealed that, whereas IL-1ß (1 ng/ml) increased islet HO-1 levels above control, islets cultured with NMMA (1 mM) in the presence of IL-1ß had significantly lower HO-1 levels, which were similar to levels in islets treated with NMMA alone (Fig. 2Go).



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Figure 4. Effects of SNP on HO-1 expression levels. A, Islets were cultured for 20 h in the absence (control) or presence of various concentrations of SNP, as indicated; B, time-dependent changes in HO-1 were determined in islets cultured for up to 18 h in the absence (control) or presence of SNP (100 µM) (SNP100). HO-1 (32 kDa) levels were determined by immunoblot with 5 µg protein per lane. Results are representative of three experiments.

 
Cobalt effects on islet insulin release
During the 20-h islet culture, insulin release into culture medium in the presence of 1 µM CoCl2 (30 ± 4 µU insulin/ml·islet) was not different from basal levels (36 ± 7 µU/ml·islet) (P > 0.05), whereas insulin release in the presence of 10 µM CoCl2 (64 ± 11 µU/ml/islet) was slightly higher than basal values (P < 0.01). Culture of islets with CoCl2 at 10 µM, but not 1 µM, also increased control insulin release, to approximately three times basal values, during a 1-h incubation in KRB buffer containing 5.5 mM glucose (Fig. 5AGo). The increase in basal insulin release in islets cultured with 10 µM CoCl2 was metabolically regulated, because release was completely inhibited in cells at 4 C and was also calcium-dependent in studies performed in calcium-free KRB buffer in the presence of EGTA (10 µM) (data not shown). NiCl2 (10 µM) did not increase basal insulin release (Fig. 5AGo).



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Figure 5. Insulin release from islets. Islets were cultured for 20 h at 5.5 mM glucose in the absence (-) or presence (+) of IL-1ß, CoCl2 (1 or 10 µM) (Co, Co1 or Co10), or NiCl2 (10 µM) (Ni or Ni10), as indicated. After culture, the islets were washed and incubated in KRB buffer in the presence of (A) 5.5 mM glucose or (B) 17 mM glucose (G17) with no other additions. Insulin release was determined after a 60-min incubation (A and B). Insulin release values (µU insulin released/ml·[10 islets) are the mean ± SE for the number of independent experiments shown at the base of each bar (for Ni10, n = 3). Significant differences were determined by one-way ANOVA and Student/Newman-Keuls multiple-comparison test. A, *, P < 0.05 vs. basal values; B, *, P < 0.01 vs. glucose-stimulated control values.

 
Total insulin content of the islets after culture with IL-1ß (1 ng/ml), or with CoCl2 (1–10 µM) in the presence or absence of IL-1ß, was not significantly different (P > 0.05) from control islets (483 ± 45 µU insulin/islet), although a higher concentration of CoCl2 (100 µM) significantly reduced (P < 0.05) total islet insulin content in the presence (330 ± 36 µU insulin/islet) and absence (294 ± 37 µU insulin/islet) of IL-1ß (1 ng/ml), compared with control. Concentrations of CoCl2 higher than 10 µM were not included in these studies because of the reduced insulin content and likelihood of interference with calcium fluxes in the ß-cell (26).

IL-1ß characteristically inhibited glucose-stimulated insulin release from islets incubated with the cytokine for 20 h (Fig. 5BGo). However, when CoCl2 was included in the 20-h culture with IL-1ß, then subsequent glucose-stimulated insulin release responses were higher than observed in islets exposed only to IL-1ß and glucose (Fig. 5BGo). The protective effect of CoCl2 was concentration-dependent, with as little as 1 µM CoCl2 affording protection of the islets from the inhibitory effect of IL-1ß on glucose-stimulated insulin release. Culture of islets with CoCl2 (10 µM) alone did not affect glucose-stimulated insulin release (Fig. 5BGo). Culture of islets with NiCl2 (10 µM), in the presence or absence of IL-1ß, did not significantly affect glucose-stimulated insulin release, compared with control values (Fig. 5BGo).

Cobalt effects on glucose metabolism
Culture of islets with IL-1ß reduced glucose oxidation by about 40% (Fig. 6Go). Culture of islets with CoCl2 (10 µM) alone did not affect glucose oxidation at 17 mM glucose (Fig. 6Go) or 5.5 mM glucose (data not shown). However, the presence of CoCl2 prevented the reduction in glucose oxidation observed with IL-1ß (Fig. 6Go). The presence of NiCl2 (10 µM) did not affect glucose oxidation, and NiCl2 did not protect the islets from the inhibitory effects of IL-1ß (Fig. 6Go).



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Figure 6. Islet glucose oxidation. Islets were cultured for 20 h in the absence (control) or presence of IL-1ß (1 ng/ml), CoCl2 (10 µM) (Co10), or NiCl2 (10 µM) (Ni10). After culture, the islets were incubated in KRB buffer containing D-[U-14C]glucose (17 mM) and the same agents as were present during culture. Glucose oxidation was determined from the production of 14CO2 during a 90-min incubation. Values are the mean ± SE for the number of independent determinations shown at the base of each bar. Significant differences were determined by one-way ANOVA and Student/Newman-Keuls multiple-comparison test. *, P < 0.01 vs. control.

 
Effects of protoporphyrins on cobalt-induced responses
ZnPP inhibits competitively HO-1 activity (6). To determine whether HO-1 activity mediated the response to CoCl2, islets were cultured in the presence of ZnPP. In islets cultured with ZnPP (1–100 µM), there seemed to be no effect of ZnPP treatment on insulin release in response to a maximal secretagogic concentration of glucose (17 mM), except that 10 µM ZnPP had a small potentiating effect (Fig. 7AGo). ZnPP (10 µM) did not affect basal insulin release at 5.5 mM glucose in either control islets, IL-1ß-treated islets, or IL-1ß plus CoCl2 (10 µm)-treated islets (data not shown). Glucose (17 mM)-stimulated insulin release in IL-1ß-treated islets was significantly inhibited and was not affected by the presence of ZnPP (10 µM) (Fig. 7BGo). However, the inhibitory response to IL-1ß was completely reversed by the presence of CoCl2 during culture (Fig. 7BGo). In contrast, ZnPP (10–100 µM) reversed the protective effect of CoCl2 in IL-1ß-treated islets on glucose-stimulated insulin release (Fig. 7BGo).



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Figure 7. Insulin release in the presence of ZnPP. Islets were cultured for 20 h in the absence (-) or presence (+) of ZnPP (1–100 µM), IL-1ß, or CoCl2 (10 µM) (Co10), as indicated. After culture, the islets were washed and incubated (60 min) in KRB buffer containing 17 mM glucose (G17), for determination of insulin release (µU insulin released/ml·10 islets). Values are the mean ± SE for the number of independent determinations shown at the base of each bar. Significant differences were determined by one-way ANOVA and Student/Newman-Keuls multiple-comparison test. *, P < 0.05 vs. G17 control.

 
Hemin has also been reported to increase HO expression and protect against IL-ß effects in islets (18). In the present study, culture of islets with hemin (0.1 mM) for 18 h did not change (P > 0.05) the basal insulin release response (135 ± 69 µU/ml·10 islets), but it did increase insulin release in response to 17 mM glucose in IL-1ß (1 ng/ml)-cultured islets (1056 ± 148 µU/ml/10 islets) (P < 0.001), compared with release from islets cultured with IL-1ß without hemin (518 ± 53 µU/ml/10 islets). The inclusion of ZnPP (50 µM) with hemin (0.1 mM) and IL-1ß (1 ng/ml) during the 18-h islet culture antagonized the hemin protective response, such that glucose (17 mM)-stimulated insulin release values (658 ± 97 µU/ml·10 islets) were not significantly different (P > 0.05) from values in islets cultured with the cytokine alone.

ZnPP did not affect expression of HO-1 in control islets or in islets treated with CoCl2 and IL-1ß (data not shown). Studies using ßHC9 cells showed that ZnPP (10 µM) accumulated in cells (0.31 ± 0.04 nmol ZnPP/106 cells) after 20 h culture.

In contrast to the inhibitory effect of ZnPP, cobalt-protoporphyrin IX (CoPP) is reported to stimulate HO-1 activity (6). In islets cultured with CoPP, there was a concentration-dependent (10–100 µM) increase in HO-1 expression, which was antagonized at the lower concentration by the presence of IL-1ß (Fig. 2Go). CoPP (10–100 µM) did not affect basal insulin release in control, IL-1ß-treated islets, or IL-1ß plus CoCl2-treated islets (Fig. 8AGo). Similarly, in CoPP (10–100 µM)-treated islets, glucose-stimulated insulin release was not affected (Fig. 8BGo). However, when CoPP was included during culture of islets with IL-1ß, then glucose-stimulated insulin release was maintained at control levels observed in the absence of cytokine (Fig. 8BGo). The presence of CoPP with CoCl2 and IL-1ß during islet culture also preserved glucose-stimulated insulin secretory responses, which were comparable with control glucose-stimulated values in the presence or absence of CoCl2 (Fig. 8BGo).



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Figure 8. CoPP effects on insulin release. Islets were cultured 20 h in the absence (-) or presence (+) of CoPP (10 or 100 µM), CoCl2 (10 µM) (Co10), and/or IL-1ß (1 ng/ml), as indicated. After culture, the islets were washed and incubated in KRB buffer in the presence of (A) 5.5 mM glucose, or (B) 17 mM glucose (G17) without other additions. Insulin release was determined after 60 min. Values for insulin release (µU insulin/ml·10 islets) are the mean ± SE for four independent determinations per treatment. Significant differences were determined by one-way ANOVA and Student/Newman-Keuls multiple-comparison test. A, *, P < 0.001 vs. values in the absence of Co10; B, *, P < 0.01 vs. all other values.

 
HO activity
To evaluate responses in HO activity, ßHC9 insulinoma cells were studied to generate sufficient bilirubin product for quantitation. ßHC9 cells responded to CoCl2 and IL-1ß with increases in HO-1 expression levels (Fig. 1CGo). When ßHC9 cells were cultured 18 h in the presence of IL-1ß, however, HO activity in cell homogenates was not increased, relative to control values (Fig. 9AGo). In contrast, CoCl2 and CoPP increased HO activity, and HO activity after treatment with CoPP and IL-1ß was significantly higher than activity in cells treated with IL-1ß alone (Fig. 9AGo). ZnPP markedly inhibited HO activity in control and CoCl2-treated cells (Fig. 9AGo).



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Figure 9. HO activity in ßHC9 cells. A, ßHC9 cells were cultured for 20 h in the absence (basal) or presence of IL-1ß (1 ng/ml), CoPP (10 µM), ZnPP (50 µM), or CoCl2 (10 µM) (Co10), as indicated; B, ßHC9 cells were cultured for 20 h with IL-1ß (1 ng/ml), washed, and homogenized. Additions to the homogenate are shown beneath the horizontal bar: CoPP (10 µM), ZnPP (50 µM), or CoCl2 (10 µM) (Co10). HO activity was determined spectrophotometrically as the production of bilirubin. Values are the mean ± SE for three or four independent determinations. Significant differences were determined by one-way ANOVA and Student/Newman-Keuls multiple-comparison test. *, P < 0.01 vs. basal; {blacktriangleup}, P < 0.01 vs. IL-1ß-treated control.

 
Cell homogenates were also studied to determine direct effects of agents on HO activity. When cells were cultured 18 h with IL-1ß, and then CoPP was added to the cell homogenate, there was an almost 3-fold increase in HO activity (Fig. 9BGo). When added directly to the cell homogenate, ZnPP inhibited HO activity, whereas CoCl2 did not affect HO activity, compared with control (Fig. 9BGo).

Cobalt effects on nitrite production
Islets cultured with IL-1ß showed a marked increase in nitrite production (Fig. 10Go). Although CoCl2 (10 µM) did not affect basal levels of nitrite, the inclusion of CoCl2 with IL-1ß during islet culture significantly reduced islet nitrite production (Fig. 10Go).



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Figure 10. Effects of CoCl2 on islet nitrite production. Islets were cultured 20 h in the absence (basal) or presence of IL-1ß (1 ng/ml) and/or CoCl2 (10 µM) (Co10). Total nitrite levels (pmol) were determined in culture medium and adjusted for micrograms of islet protein per sample. Values are the mean ± SE for the number of determinations shown at the base of each bar. Significant differences were determined by one-way ANOVA and Student/Newman-Keuls multiple-comparison test.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This is the first study to demonstrate that treatment with cobalt antagonizes the effects of IL-1ß in isolated islets. IL-1ß has been characterized as inducing iNOS and apoptosis, increasing levels of NO, nitrosylating key metabolic enzymes, and inhibiting insulin release in pancreatic islets cultured in vitro or in insulinoma cells (13, 23, 27, 28, 29, 30). iNOS increases in islets of diabetic mice (31) and may play a role in islet ß-cell destruction in diabetes mellitus (32). Agents previously demonstrated to antagonize the IL-1ß-induced inhibition of insulin release include inhibitors of iNOS (12, 27, 33) and nuclear factor-{kappa}B (34). In the present study, CoCl2 also protected against the effects of IL-1ß in islets, and one major pathway associated with cobalt exposure of cells is induction of HO-1. It has been previously reported that CoCl2 effects on cells do not include induction of the expression of the hsp70 gene (35), and our results confirm that changes in hsp70 do not parallel changes in HO-1 in response to CoCl2 or IL-1ß at the concentrations used in this study. It was previously reported that IL-1ß increased hsp70 and HO-1 levels in isolated ß-cells after 24 h culture (36). In the present study, hsp70 was also clearly expressed in islets, although it was not evident that IL-1ß or CoCl2 enhanced those levels. The high basal levels of hsp70 in the present study, as also reported for isolated ß-cells (36), may have minimized the changes detectable with the stimuli, but the results suggest that HO-1 responses and effects are independent of changes in ß-cell hsp70 levels. Co2+ can also antagonize calcium fluxes in cells, but this occurs at high (millimolar) concentrations of the cation (26) and results in inhibition, rather than stimulation, of hormone release. Because calcium influx is a major component of glucose-stimulated insulin release (37) and secretory responses were calcium-dependent in the present study, it does not seem that the protective effects of CoCl2 during IL-1ß exposure can be attributed to reduced calcium fluxes.

HO is found in pancreatic islets as both the inducible HO-1 and constitutive HO-2 enzymes (3, 4, 5). The increase in HO-1 in islets during short-term culture confirms the observation that rat islet HO increased dramatically, compared with human islet HO, during 5–8 days culture (5). A putative role for HO-2 in glucagon and insulin secretion has been proposed, especially in regard to the generation of carbon monoxide (4). In the present study, the increased basal insulin release in islets treated with CoCl2 may be related to enhanced HO-1 expression, because NiCl2 failed to mimic the HO-1 response or evoke insulin secretion in this study. Whether or not carbon monoxide mediates HO-1 and CoCl2 effects on insulin secretion, as suggested by other investigators regarding HO-2 (4), is not known.

Exposure of islets to CoCl2 induced HO-1 expression in a concentration- and time-dependent manner. Previous reports indicate that HO-1 expression is increased within a few hours after a stimulus is applied, although levels of HO-1 remain elevated for several days (6, 38). In the present study, CoCl2 increased islet HO-1 levels within 4 h, and maximum expression was observed within 8 h; a similar but augmented response was observed when IL-1ß was combined with CoCl2. The reduced expression level of HO-1 in islets treated with IL-1ß and CoCl2vs. CoCl2 alone suggests that IL-1ß has effects on protein biosynthesis. A similar inhibitory effect of IL-1ß on low-concentration CoPP-induced HO-1 expression was observed. However, a higher concentration of CoPP overcame the inhibitory effect of IL-1ß, suggesting that islets retained their protein biosynthetic capacity.

Treatment of islets with IL-1ß alone also enhanced HO-1 levels above control to a small extent, in agreement with previous reports (18, 33, 36, 39). In islets, NO seems to mediate the HO-1 response to IL-1ß, because HO-1 expression was increased in a time- and concentration-dependent manner in response to the NO-generating agent SNP, as reported for endothelial cells (25). Moreover, the inhibition of iNOS by NMMA was associated with a reduction in HO-1 expression in response to IL-1ß, supporting the hypothesis that NO mediates the IL-1ß-induced increase in HO-1. Because NO increases free intracellular heme (40), the increased HO-1 expression in response to NO may be a response to protect cells from free heme through metabolism to biliverdin and bilirubin. The question arises as to why IL-1ß-induced increases in HO-1 are not protective against the cytokine? One explanation may be that IL-1ß did not increase HO-1 levels until late after initial exposure to the cytokine, perhaps too late to exert a protective effect. A second explanation may be that although IL-1ß modestly increases HO-1 protein levels, the HO-1 activity may not parallel the change in expression. HO-1 activity is inhibited by NO and PG E2 (41, 42), both of which are induced during cell exposure to cytokines in islets and other cell types (43, 44). Thus, impaired HO-1 activity may not be sufficient to protect the ß-cell from the toxic effects of IL-1ß. The results show that CoCl2 treatment markedly reduces the time for expression of HO-1 and augments the HO-1 levels in IL-1ß-treated islets, compared with islets treated with IL-1ß alone, which may contribute to the protective response observed with CoCl2. The failure of NiCl2 to induce HO-1 in islets or to protect the secretory response from the effects of IL-1ß illustrates the specificity of the Co2+ response. Although Ni2+ is a heavy metal, it is known that tissues differ greatly in their responsivity to different metals, regarding HO-1 induction (40).

The specificity of the protective response of cells to CoCl2 was demonstrated also in the maintenance of glucose oxidation in IL-1ß-treated islets, whereas a similar concentration of NiCl2 proved ineffective. IL-1ß has been reported previously to inhibit glucose oxidation (23, 45, 46), apparently because of generation of NO. Glucose-stimulated insulin release is dependent upon ATP production and regulation of ATP-sensitive K+-channels, which regulate cell depolarization, calcium influx through voltage-dependent calcium channels, and secretion (37). NO causes ADP-ribosylation and nitrosylation of certain enzymes in the glucose-oxidative pathway and mitochondrial enzymes (13, 40), in addition to reducing glucokinase messenger RNA (mRNA) and protein levels (46). The protective actions of CoCl2 on insulin release may be mediated, in part, through preservation of glucose metabolism and energy production as a result of the inhibition of iNOS activity and reduced NO production. CoCl2-treated islets showed a significant reduction in total nitrite levels, which may be protective against the effects of IL-1ß. CoCl2 effects on nitrite production may be mediated through changes in iNOS activity, because one of the products of HO-1, carbon monoxide, inhibits iNOS by binding to its heme moiety (47, 48). HO-1 induction and increased heme metabolism may also limit iNOS activity caused by heme restriction. Because NO is a potent stimulator of soluble guanylyl cyclase, it does not seem likely that any additional cyclic GMP formed as a result of carbon monoxide stimulation of guanylyl cyclase (49, 50) contributed to insulin release.

Additional evidence for the involvement of HO-1 in the CoCl2 effect on glucose-stimulated insulin secretion in IL-1ß-treated islets was the concentration-dependent reversal of the protective effect by an inhibitor of HO-1, ZnPP (6). Because ZnPP-treatment did not affect glucose-stimulated insulin release in cells treated or not with IL-1ß, and ZnPP did not affect HO-1 expression, the antagonistic response would seem to be modulated through HO-1 activity changes. Previously, ZnPP was reported to inhibit HO-2 activity and carbon monoxide production in rat islets and to inhibit secretory responses induced by glucose and hemin, a substrate and inducer of HO (4). After overnight culture of islets with ZnPP, we failed to observe the small inhibition of glucose-stimulated insulin release that was reported after acute treatment of freshly isolated islets with ZnPP (4), although the reason for this is not evident. However, the present results confirm that hemin protects against the effects of IL-1ß on insulin release (18) and extend the observations to include ZnPP antagonism of the hemin protective response, probably caused by inhibition of HO activity.

Selectivity of the ZnPP response in the present study was demonstrated when CoPP, which increased HO-1 expression in islets, in agreement with the powerful inducer effects reported for other tissues (6), did not antagonize the CoCl2 response. On the contrary, CoPP increased the expression of HO-1 in islets and mimicked the islet response to CoCl2, in terms of protecting the cells from the inhibitory effects of IL-1ß. The ability of CoCl2 to induce HO-1 in rat liver requires conversion of Co2+ into CoPP (51); however, it is not known whether this conversion is responsible for CoCl2 HO-1-inducing activity in islets. Although certain of the metal-protoporphyrin inhibitors are selective for HO-1, they can also inhibit iNOS (52). However, if ZnPP inhibited iNOS in islets, then it would be expected that the islets would be protected against the effects of IL-1ß, and this did not occur. However, a inhibitory effect of CoPP on iNOS cannot be ruled out, and the interference by CoPP in the Greiss reaction for nitrite determination prevented assessment of this possibility. It is also possible that Co2+ or CoPP interfere with NO actions in cells or modify IL-1ß responses. ZnPP has been reported to reduce IL-1ß responses (53). However, the concentration of ZnPP chosen in the present study did not significantly affect IL-1ß responses in the absence of inducers of HO-1, suggesting the agent did not directly affect the cytokine response.

One of the inconsistencies in our results, regarding the hypothesis that HO-1 induction mediates protective responses against IL-1ß in islets, was the observation that HO-1 levels at a low concentration of CoPP in IL-1ß-treated islets was similar to levels in islets treated with IL-1ß alone, and yet insulin release responses were protected. Investigation of the HO activity in ßHC9 cells revealed a possible explanation. As expected, HO activity increased in ßHC9 cells cultured with CoCl2 or CoPP, and the activity was inhibited with ZnPP present during culture. And, although IL-1ß increased HO-1 expression in ßHC9 cells, HO activity was not different from basal, suggesting that an endogenous inhibitor of HO activity may be produced in ß-cells. When HO activity was assessed in homogenates from cells cultured with IL-1ß, a direct stimulatory effect of CoPP on HO was observed. CoCl2 did not mimic the direct effect of CoPP to activate the enzyme, and addition of ZnPP inhibited the HO activity. Thus, a possible explanation for the ability of low levels of CoPP to protect the ß-cells from the effects of IL-1ß is that CoPP directly activated HO activity, even when expression levels of HO were relatively low. In addition, CoPP activation of HO-2 in islets may contribute to increased insulin release.

In summary, these results suggest that the induction and/or activation of HO, in response to Co2+, CoPP, or hemin in islet cells, provides a protective mechanism against the actions of IL-1ß and preserves the glucose-stimulated insulin secretory response and glucose metabolism. The mechanism by which CoCl2 induces HO-1 expression is not known, although studies have described effects of oxidant stress (54), CoPP generation (51), and activation of distal response elements in HO-1 gene regulation (10) as mediating Co2+ responses. Moreover, the results indicate that inhibition of iNOS activity, perhaps mediated through the HO-1-generated product carbon monoxide, plays a role in the protective effect of Co2+ on cytokine-treated islet cells. It is also possible that the other products of HO activity, the antioxidant bilirubin (1), or the iron produced that can consume NO (55), have a protective role in the actions of Co2+ and HO-1 expression.


    Acknowledgments
 
The expert technical assistance of Jill Platten is appreciated.


    Footnotes
 
1 This work was supported by NIH Grant DK-25705 (to S.G.L.). Back

2 Portions of this research were completed in partial fulfillment of the degree of Doctor of Philosophy. Back

Received April 6, 1998.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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